In brain, intercellular communication, nutrient and metabolite trafficking, and the delivery of drugs takes place in the extracellular space (ECS). Diffusion, the major transport mechanism mediating these processes, is governed by two structural parameters of the ECS, tortuosity and volume fraction. The tortuosity (?) represents the hindrance imposed on the diffusing molecules by the tissue in comparison with an obstacle-free medium, whereas the volume fraction (a) is the proportion of tissue volume occupied by the ECS. A fundamental question remains unanswered: what hinders molecules traveling through the brain? In healthy brain, ? extracted from the diffusion of small ECS markers is about 1.6 but increases to 1.9 in pathologies where cells swell. It had been thought that ? can be explained by circumnavigation of markers around cells. However, ? derived theoretically or obtained from simulations in media composed of convex cells exhibits an upper limit of about 1.23. Obviously, some other significant factor determines ? in the brain. The central hypothesis of this proposal is that the ECS contains pocket-like microdomains that can significantly slow down the diffusion process, and that these microdomains are formed and regulated by glia. The iontophoresis-based tetramethylammonium (TMA) method and integrative optical imaging (IOI) will quantify diffusion of TMA+ and fluorescent macromolecules, respectively. Rat neocortical slices will be the main preparation but some experiments will examine the cerebellum, hypothalamus or brainstem because of the unique morphological features of these regions. There are three specific aims. Aim 1. Establish that pocket-like microdomains hinder diffusion in brain ECS. The ECS structure will be altered by background macromolecules that fill the pockets and ? will be measured. Trapping of macromolecules will be characterized by the IOI and electron microscopy will localize the entrapment sites. Aim 2. Show that glia form and regulate microdomains. Tortuosity will be measured in the cerebellum where glial wrappings are abundant, in the hypothalamus where withdrawal of glial processes from the supraoptic nucleus will be induced pharmacologically, and in the neocortex where glial toxins will swell glia. Aim 3. Measure diffusion within a microdomain. Diffusion will be measured within a microdomain model, the giant calyx of Held synapse in the brainstem. Computer simulations of diffusion will complement experimental work on Aims 1 and 3. This proposal is focused on basic research with major implications for transport of substances in the nervous system. It identifies a novel role for glia in regulation of diffusion in the ECS.